amplicon sequencing of HEK293T cells: HyperDriveCas9_HEK_site4_off20_rep3
HEK293T cells were cultured at 37C under humidified 95% air/5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing glucose (4.5 g/l), 1 mM sodium pyruvate, 2 mM glutamine and 10% fetal bovine serum (FBS). Cells were seeded at 60% confluence in 24-well plates and transfected after 24 h with 500 ng of DNA in OptiMEM media (Gibco, Life Technologies) using a 1:1 mix of two transfection reagents (Lipofectamine LTX Reagent with Plus Reagent, Invitrogen, and FuGene HD, Promega). 72 h after transfection the cells were trypsinized and the cell pellets lysed for DNA extraction using the KAPA Express Extract Kit, according to the manufacturers instructions (Sigma-Aldrich). Amplicons were generated using primers flanking the gRNA and incorporating Illumina adaptor sequences.